Pharmacological effects of caffeine on ventilation in adult zebrafish under free-swimming conditions

The zebrafish is widely used as a model in biological studies. In particular, the heart rate and cortisol levels of zebrafish are commonly measured to elucidate the pharmacological effects of chemical substances. Meanwhile, although ventilation is also an important physiological index reflecting emotion-like states, few studies have evaluated the effects of chemicals on ventilation in adult zebrafish. In this study, we assessed whether it is possible to evaluate the pharmacological effects elicited by caffeine in adult zebrafish under free-swimming conditions. We measured the ventilation in adult zebrafish exposed to multiple concentrations of caffeine under restraint and free-swimming conditions and evaluated the pharmacological effects of caffeine using linear mixed model analysis. In addition, results of electrocardiogram analysis and swimming speeds were compared with those in previous reports to ensure that an appropriate dose of caffeine was administered. Under restraint conditions, caffeine significantly decreased heart rate and increased ventilation in a concentration-dependent manner. Under free-swimming conditions, the ventilation rate significantly increased with increasing caffeine concentration. These results indicate that the pharmacological effects elicited by chemicals on ventilation can be evaluated in free-swimming zebrafish.


S1. Estimation of individual number
We used a simulation-based linear mixed model power analysis to estimate the sample sizes for experiments 1 . This analysis employed peak frequencies of ventilation measured under free-swimming conditions described in the main text. Figure S1 shows the power curve.
According to the figure, the power to detect an assumed slope of 0.2 for logarithmic caffeine concentration exceeds 80% for five individuals and five repeated measures at each concentration. Because traditionally, 80% power is considered adequate 2 , we decided to select five individuals for each concentration.

S2. The relationships between the ventilatory signal and gill movement under restraint conditions
We compared the frequencies of gill movements and ventilatory signals. The ventilatory signal and the gill movement were measured under restraint conditions. The measurement system used in the restraint condition was modified to simultaneously measure ventilatory signals and gill movements. Figure. S2(a) shows the measurement system consisting of a fixture, measurement tank, high-speed camera, bioamplifier, and three electrodes. The fixture was made up of a conductive urethane sponge hollowed in the shape of a fish and wrapped in transparent vinyl to hold the fish in place. The fixture was fixed to the bottom of the measurement tank using hook and loop fasteners. An Ag-AgCl electrode was placed on the fixture close to the gill to measure the ventilatory signal, and two Ag-AgCl electrodes were placed on the bottom of the tank as the reference electrodes. The measured ventilatory signal was sampled at Hz using a digital bioamplifier (EEG-1200, Nihon Kohden, Tokyo, Japan). A high-speed camera (Ace USB3.0, Basler AG, Ahrensburg, Germany) was installed above the measurement tank to capture images of the zebrafish head at fps to measure gill movement.
The gill movements were measured using the same procedure as described in the main text.
Polygonal regions of interest (ROIs) were set around the gill area of the captured video image, as shown in Fig. S2(b). The mean brightness in the ROI was calculated for each frame, and the obtained time-series waveform was defined as the gill movement. Figure S3 illustrates the experimental procedure. A mixture of dechlorinated water and 2phenoxyethanol (for anesthesia) was prepared. 400 mL of the mixture was added to both the preparation and the measurement tanks. The concentration of 2-phenoxyethanol was μL/L. A randomly chosen individual was transferred from the breeding tank to the preparation tank and exposed to 2-phenoxyethanol for 15 min. After the fish was transferred from the preparation tank into the measurement tank, it was restrained in the fixture, as shown in Fig. S2(a). Finally, the ventilatory signals and gill movements were recorded for 120 s.
The correlation between peak frequencies of gill movements and that of ventilatory signals was assessed. First, each signal was filtered using an -order Butterworth bandpass filter. The low and high cutoff frequencies ( , ) were set based on the values reported in a previous study 3 . Each filtered time-series signal was divided into s segments, and the amplitudes were standardized to a normal distribution with a mean of 1 and a variance of 0. The peak frequencies were calculated by estimating the power spectral density of each signal at each time segment ( s) using the Burg's method applied to aorder autoregressive model (AR).
During the experiment, the room and water temperature were maintained at 27 and 29℃, respectively. The concentration of 2-phenoxyethanol was set at = 260 μL/L. The ventilatory signal and gill movement of one individual were measured. The frame rate of the high-speed camera was = 100 fps, and the sampling frequency of the ventilatory signals was = 1 kHz. The order of the Butterworth filter was set to = 3, and the cutoff frequency of the ventilatory signal and gill movement was set to = 1 Hz, = 10 Hz. The order of the AR model was determined using the AIC at each = 5 s segment in the range of = 10 -50. Figure S4 shows the scatter plot between the peak frequencies of the ventilatory signals and gill movements. This analysis demonstrated that the peak frequencies are highly correlated ( = 0.981, < 0.001), indicating correspondence between the ventilatory signals and gill movements.